Methods of Liver Disease Treatment

ABSTRACT

Methods for treatment of chronic liver disease and reducing liver fibrosis are provided. These treatments may be achieved using a medical food composition. The medical food is configured specifically for those having chronic liver disease to provide for specific nutritional requirements caused by the chronic liver disease. Testing of the treated patient allows for tracking of progress and to determine if the liver fibrosis is reduced.

BACKGROUND

The present invention relates to medical food compositions. Moreparticularly, the present invention relates to a medical food forpatients with Chronic Liver Disease specifically formulated to fulfillspecific, distinctive dietary requirements of patients with chronicliver disease. In some embodiments, the medical food contemplated hereinmay reverse liver fibrosis.

Chronic Liver Disease (CLD) continues to rise in countries worldwide andit is a growing problem in the US in particular. The spectrum of CLDinclude, but is not limited to the predominate hepaticdiseases—hepatitis C (HCV) and hepatitis B (HBV) viruses, non-alcoholicfatty liver disease (NAFLD) with associated non-alcoholicsteatohepatitis (NASH), and alcoholic liver disease (ALD). All of thesechronic liver conditions may lead to cirrhosis and hepatocellularcarcinoma (HCC). While new HCV antiviral drugs are effective ateliminating the virus in 90% of HCV-infected patients that receive them,the expense of these new drugs has limited availability in the shortterm. Effective prophylactic HBV vaccines have reduced the risk of HBVin this country, but HBV is still a worldwide epidemic and anti-viralmedications are only prescribed in chronic at-risk HBV cases. Further,while we have made medical advances in HCV antivirals and HBV vaccines,NAFLD and NASH are on the rise in this country. The rampant epidemic ofobesity has led to significant rates of NAFLD affecting 17% to 33% ofindividuals in the United States. It is estimated that six millionpeople have progressed to NASH. It is currently projected that NASHpatients will soon overtake HCV patients in the number of livertransplantations performed every year.

CLD has been the subject of extensive studies over the decades that addto our collective knowledge of hepatic fibrogenesis, liver disease andthe various means by which CLD disrupts metabolism. CS Leiber was apioneer in these areas, performing many animal studies involving ALD,steatosis, cirrhosis and the administration of phosphatidylcholine (PC)and/or SAMe to correct CLD-associated metabolic disruptions and hepaticfibrosis.

In a historical context, ten years ago valuable new types of studiescalled metabolomic studies were begun and started being reported in theliterature. Since that time metabolomic studies have become establishedas mainstream science. Metabolomics involves a comparative analysis ofglobal metabolic profiles between two or more samples. Extensivemetabolomic studies have been performed on hepatobiliary diseases overthe last ten years. Metabolomic studies confirmed the findings of pastCLD studies documenting various disruptions of metabolism, including GSHmetabolism, PC metabolism, SAMe/homocysteine metabolism, one-carbonmetabolism, redox homeostasis, etc. seen in all patients with CLD.

Metabolomic studies have also provided new and valuable insights intoCLD pathology, especially because these studies specifically identifymetabolic alterations and core metabolic phenotype (CMP) changesassociated with CLD. Metabolomics involve high-throughput analyticchemistry combined with multivariate data analysis to compile anunbiased, and often extensive profile of small metabolites from varioussamples including animal models, in-vitro hepatocytes, live humansamples, serum, plasma, etc. The focus of Metabolomic studies on theliver is at least partly due to the fact that no other organ in the bodyhas nearly as much metabolic activity as the liver, so it is an obviouschoice as an organ to study changes in the metabolome associated withdisease states, in this case CLD.

The hepatic metabolome consists of a very complex collection ofsmall-molecule (<1.5 kDa) lipid and water-soluble metabolites thatinteract in complex ways. The flux of these metabolites providesinformation about genomic, proteomic and transcriptomic activity thatcorrelate with both normal and disease-altered hepatic metabolism.Extensive Metabolomic analyses on CLD samples over the last decade haveidentified specific metabolic/phenotypic alterations associated withvarious forms of hepatobiliary disease states. Not surprising, Leiberidentified many of these same CLD-disrupted pathways in his manyhistoric liver disease studies.

Strikingly, hepatic metabolic alterations to the phenotype associatedwith CLD all exhibit a similar, almost identical global profile. Inother words, CLD progresses in similar patterns defined by similarepigenetic alterations in metabolic activity, regardless of the etiologyof the disease. The profile of these combined alterations make up aspecific “Core Metabolic Phenotype” (CMP) in patients with CLD.According to Beyoglu et al (cited herein),

-   -   “Whether provoked by obesity and diabetes, alcohol use or        oncogenic viruses, the liver develops a core metabolomic        phenotype (CMP) that involves dysregulation of bile acid and        phospholipid homeostasis. The CMP commences at the transition        between the healthy liver (Phase 0) and NAFLD/NASH, ALD or viral        hepatitis (Phase 1). This CMP is maintained in the presence or        absence of cirrhosis (Phase 2) and whether or not either HCC or    -   CCA (Phase 3) develops. Inflammatory signaling in the liver        triggers the appearance of the CMP. Many other metabolomic        markers distinguish between Phases 0, 1, 2 and 3. A metabolic        remodeling in HCC has been described but metabolomic data from        all four Phases demonstrate that the Warburg shift from        mitochondrial respiration to cytosolic glycolysis foreshadows        HCC and may occur as early as Phase 1. The metabolic remodeling        also involves an upregulation of fatty acid β-oxidation, also        beginning in Phase 1. The storage of triglycerides in fatty        liver provides high energy-yielding substrates for Phases 2 and        3 of liver pathology.”

A comprehensive review of metabolomics studies show that all forms ofCLD similarly present the following CMP-associated changes:

extreme oxidative stress,

increased β-oxidation of fatty acids,

a shift from aerobic respiration to anaerobic glycolysis,

increased glutathione/cysteine/thiol cycling,

dysregulation of phospholipid and bile acid homeostasis and

increased storage of cytosolic fatty acids and triacylglycerides infatty liver.

Most of these CMP-associated metabolic alterations involve up-regulatedbiosynthetic pathways whose end-products experience greater utilizationand consumption. CLD-induced up-regulated biosynthetic pathwaysexperience greater flux of metabolites, while the biosyntheticend-products of these pathways experience decreased availability ordepletion due to greater utilization. Increased cycling ofCMP-upregulated metabolites, therefore, is regarded as increasedmetabolic demand associated with CLD.

In other words, CLD causes alterations to the phenotype of the host byaltering and up-regulating specific metabolic/biosynthetic pathways. Thecombined metabolic alterations induced by CLD create increased metabolicdemand and utilization for CMP-upregulated metabolites and nutrients.Increased metabolic demand translates into increased nutritionalrequirements for these metabolites. Therefore, increased metabolicdemands associated with CMP-upregulated metabolites define the newdistinctive nutritional requirements of CLD patients.

Identification of these CLD-altered metabolic/biosynthetic pathways isnecessary to develop a science-based medical food. The term medicalfood, as defined in section 5(b) of the Orphan Drug Act (21 U.S.C. 360ee(b) (3)) is “a food which is formulated to be consumed or administeredenterally under the supervision of a physician and which is intended forthe specific dietary management of a disease or condition for whichdistinctive nutritional requirements, based on recognized scientificprinciples, are established by medical evaluation.”

Therefore, what is needed is a medical food for CLD patients thatsupplies appropriate amounts of specific nutrients for which CLDpatients experience greater demand and utilization. This medical foodfor CLD patients will support their new distinctive nutritionalrequirements.

SUMMARY

The subject matter of this application may involve, in some cases,interrelated products, alternative solutions to a particular problem,and/or a plurality of different uses of a single system or article.

In one aspect, an ingestible composition is provided. The compositionmay comprise a Cysteine-providing ingredient in a range of 500-5,000 mg;Polyenylphosphatidylcholine in a range of 500-10,000 mg; and AlphaLipoic Acid in a range of 200-2,500 mg. This composition may be dividedinto individual serving sizes for administration. In a further aspect,the composition may further comprise at least one of L-Lysine;L-Arginine; Vitamin C; N-Acetyl L-Carnitine; Betaine HCl; L-Glutamate;Turmeric; Proanthocyanidins; Nigella sativa; Pantothenic acid;Magnesium; Vitamin E; Cynara scolymus; L-Glycine; Vitamin B1; VitaminB2; Ubiquinol; Piper cubeba; Vitamin B3; Vitamin B6; Zinc; Vitamin D3;Folate; Vitamin B12; Selenium; Vitamin A, Vitamin K, and Biotin. Thecomposition may also optionally include Cannabidiol.

In another aspect, medical food composition selected to aid in areduction or reversal of liver fibrosis is provided. The composition maycomprise a Cysteine-providing ingredient in a range of 500-5,000 mg;Polyenylphosphatidylcholine in a range of 500-10,000 mg; and AlphaLipoic Acid in a range of 200-2,500 mg. This composition may be dividedinto individual serving sizes for administration. In a further aspect,the composition may further comprise at least one of L-Lysine;L-Arginine; Vitamin C; N-Acetyl L-Carnitine; Betaine HCl; L-Glutamate;Turmeric; Proanthocyanidins; Nigella sativa; Pantothenic acid;Magnesium; Vitamin E; Cynara scolymus; L-Glycine; Vitamin B1; VitaminB2; Ubiquinol; Piper cubeba; Vitamin B3; Vitamin B6; Zinc; Vitamin D3;Folate; Vitamin B12; Selenium; Vitamin A, Vitamin K, and Biotin. Thecomposition may also optionally include Cannabidiol.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 provides a flow chart of an embodiment of use of the medical foodof the present invention.

DETAILED DESCRIPTION

The present medical food composition accomplishes the need of providingCLD patients with their new distinctive nutritional requirements. Thepresent invention is a new medical food intended to fulfill the specificdietary requirements of patients with CLD for which distinctivenutritional requirements, based on established epigenetic, metabolomicand other types of scientific research, are established.

The composition of the present invention is referred to herein as a“medical food” or the “medical food of the present invention.” This termis intended to apply to the composition discussed for the treatmentcontemplated. However, it should be understood that the composition ofthe present invention may be used as a dietary supplement formula, aswell as a drug (such as an FDA approved drug) formula.

The complex list of metabolites that are required to stabilize theseinterlinked up-regulated metabolic pathways can only be provided by astrategic and comprehensive medical food. The complex balance betweenthe medical food's numerous nutrients and The medical food's requirementof continuous administration three times per day could not be reasonablyprovided by simple alterations of diet alone.

In a particular embodiment, the medical food may be administered orallyin divided doses of eight capsules taken three times a day (morning,afternoon, and evening) for a total of twenty four capsules daily.However, it should be understood that the formulation may also besupplied as a liquid drink, tablet, powder, gel, emulsion, micelles,liposomes, and the like.

Further, CLD is not a nutrient deficiency disease like scurvy orpellagra, in which the supplementation of a particular deficientnutrient or dietary supplement may correct symptoms caused bydeficiencies of a person's normal nutritional requirements. Similarly,ALD does not simply alter the metabolism of the patient while he isdrinking. Rather the abuse of alcohol may change the patient'smetabolism in the long-term and alter his nutritional requirements inways that require fulfillment even after the patient has quit drinking.The medical food is designed to address and correct the results ofunfulfilled nutritional requirements in CLD patients.

The medical food satisfies the new distinctive nutritional requirementsof CLD patients with a comprehensive list of required metabolitesdesigned to fortify and balance all CMP interlinked and up-regulatedmetabolic pathways associated with patients with CLD. Research showsthat these linked CMP-upregulated biosynthetic pathways experienceconcurrent increased demand and utilization. Therefore, a comprehensiveand balanced approach is necessary in order to fulfill the newdistinctive nutritional requirements of CLD patients.

Each medical food daily dose is split into three separateadministrations in order to satisfy the constant increased nutritionalrequirements of CLD patients. The medical food's 3× daily administrationis designed to achieve optimal periods of homeostasis in CMP-disruptedsystems for as long as possible throughout the day. Once or twice dailyadministrations of medical food will not be as effective as the presentinvention's three-time-daily administration, even with the same totaldaily amount This is because the medical food of the present inventionis designed to provide increased nutrient delivery to the cells of CLDpatients in an optimal manner throughout the day, and a three-time-dailyadministration is necessary to achieve this purpose.

The medical food has identified all known metabolic pathwaysdemonstrating CMP up-regulation in CLD. Fulfilling the new distinctivenutritional requirements for CLD patients is accomplished by supplyingthe necessary balanced amounts of critical nutrients and metabolites forincreased flux into CMP-associated up-regulated pathways.

The present invention is a medical food that fulfills new distinctivenutrient requirements for patients with CLD. Medical foods are not drugsand by regulation may contain only GRAS (Generally Recognized As Safe)ingredients. Every ingredient in the medical food is classified as GRASby the FDA. Further, each ingredient in the medical food formula is inproper balance and proportion for safe administration. The medical foodGRAS ingredients are required to provide nutritionally balanced supportfor CMP up-regulated metabolic/biosynthetic/antioxidant pathways andhomeostatic activity in CLD patients.

The medical food is not intended to treat or cure CLD. Rather, themedical food for chronic liver disease of the present invention isintended to satisfy the new distinctive nutritional requirements of CLDpatients. Our intention is for the present invention's status as amedical food to be satisfied by even the FDA's narrowest interpretationof the statute. In the present clinical setting, there is evidence of aneed for such a science-based medical food for CLD patients.

Core Metabolic Phenotype Comprehensively Identifies CLD-AlteredMetabolic/Biosynthetic Pathways

As shown below, all CLD exhibit very similar changes to core metabolicphenotype (CMP), regardless of CLD etiology. Specific studies willdemonstrate that the same list of CMP-associated metabolic alterationsoccur in each and every type of CLD. The purpose of this exercise is toestablish that due to the very similar nature of these CLD-inducedmetabolic disruptions, all patients with CLD experience the samedisrupted metabolic pathways. These disrupted metabolic pathways definethe new distinctive nutritional requirements for patients with CLD. Itis therefore appropriate to fulfill the new distinctive nutritionalrequirements of CLD patients by providing the same medical food formula,because the medical food of the present invention addresses the samedisrupted metabolic pathways present in every form of CLD, regardless ofthe etiology. The medical food accomplishes this by providing a balancedand comprehensive medical food formula with scientific justification andup-coming human clinical trials.

CMP is associated with the following global changes in the metabolome ofCLD patients:

-   -   Dis-regulation of phospholipid metabolism, as well as        cholesterol and bile acid homeostasis,    -   Increased storage of cytosolic triglycerides and fatty acids        accompanied with altered β-oxidation of fatty acids,    -   A shift from aerobic respiration to anaerobic glycolysis,    -   Increased utilization of thiol-containing metabolites including        increased glutathione/cysteine cycling,

CMP-associated metabolic changes listed above combine to create extremeoxidative stress, which the evidence has been well documented in allCLD. Therefore, as stated above, the rest of Part A will be dedicated toestablishing the new CMP-associated distinctive nutritional requirementsof CLD patients. The discussion about how to fulfill the new distinctivenutritional requirements of CLD patients will take place in Part B,below.

1. CMP is associated with Disruption of Phospholipid, Cholesterol andBile Acid Homeostasis

Phosphatidylcholine (PC) Homeostasis

Many Metabolomic studies have focused on CMP-associated changes in thelipidome of CLD patients. It has long been known that lipid metabolismexperiences widespread disruption in patients with CLD and that thesemetabolic changes are associated with distinct changes in specificmetabolic biomarkers. Of particular interest to metabolomic researchershas been CMP-associated disruption of PC homeostasis. It has beendemonstrated in a variety of studies that all types of CLD patientsexperience decreased PC availability due to increased utilization and/orimpaired synthesis in patients with CLD.

For example, a meta-analysis that examined a large body of metabolomichepatobiliary studies involving patients with all types of CLDdocumented that lysophosphatidylcholine species (LPCs) were universallydepressed while bile acids were found elevated in all forms of CLD.Thus, the study concluded that “It would appear that depressed LPCs andelevated bile acids in serum represent a phenotype of hepatitis andcirrhosis independent of etiological origin . . . .

Cholesterol and Bile Acid Homeostasis

Similarly, CLD-associated CMP changes cause elevated levels ofcholesterol and bile acids. Increased cholesterol infiltration of themitochondrial membrane has been associated with mitochondrial GSHdepletion. Bile acids are created from cholesterol and bile acids arefound elevated in patients with CLD.

Glycochenodeoxycholic acid (GCDCA) is the main toxic component of bileacid and elevated GCDCA has been shown to be significantly associatedwith decreased mitofusin 2 (Mfn2) gene expression. Decreased Mfn2 isassociated with many mitochondrial-related diseases. This wasdemonstrated in a study conducted by Chen et al in which normal humanhepatocytes induced by GCDCA showed significant decreased in Mfn2activity resulting in mitochondrial damage. However, this mitochondrialdamage was reversed in the lab by stimulating overexpression of Mfn2.

1a. Non-Alcoholic Fatty Liver Disease (NAFLD)—Disruption of PC,Cholesterol and Bile Acid Homeostasis

PC Homeostasis

NAFLD exhibits CMP-associated disruption of PC biosynthetic pathways. Astudy comparing human steatotic livers versus non-steatotic liversshowed elevated PC metabolites in steatotic livers, indicating increasedactivity in the PC biosynthetic pathway. Another research studydocumented rodents who were fed diets that induced fatty liverexperienced up-regulated utilization of PC, choline, betaine, andtrimethylamine N-oxide. Other human and animal studies have shown PCdisruption in NAFLD.

Increased catabolism of PC and phosphatidylethanolamine (PE) species inthe liver by phospholipases A1 and A2 release free fatty acids thatrequire β-oxidation in the mitochondria. If these hepatic fatty acidsare not adequately oxidized by β-oxidation, then the unused fatty acidswill be repackaged in the liver as hepatic triacylglycerols, creatinghepatic steatosis (NAFLD) and setting the stage for NASH.

Metabolomic studies show that CMP-associated disruption of PChomeostasis and elevated triacylglycerol production are not simply anintracellular accumulation of fat in the liver but rather a large-scaletranslocation of lipid stores. Decreased PC availability and increasedPC utilization may contribute to increased cholesterol and fatty acidinfiltration into cellular and mitochondrial membranes, which may inturn affect mitochondrial glutathione (GSH) transport

Cholesterol and Bile Acid Homeostasis

In human and animal studies NAFLD causes an increase in lipid species inthe liver and serum/plasma, including cholesterol esters and variousbile salts. However, NAFLD is marked by less hepatic inflammation, andtherefore less bile acids disruption than NASH.

1b. Non-Alcoholic Steatohepatitis (NASH)—PC, Cholesterol and Bile AcidHomeostasis

PC Homeostasis

NASH is defined as advanced-stage NAFLD accompanied by high inflammatoryactivity. As much as 67% -80% of NAFLD patients may remain as benignfatty liver with minimal progression to cirrhosis and normal mortalityrates compared to the general population. However, approximately 20%-33%of NAFLD patients may progress to NASH.

In both NASH and in NAFLD, triacylglycerols and several fatty acids areshown to be elevated in the liver, while various other fatty acids andlysophosphatidylcholine (LPC) show decreased levels in plasma. Metabolicstudies of NAFLD and NASH showed global changes to lipid profiles inboth diseases with only a few differences between them—a comparativeanalysis showed only three phospholipid species with significant changesin serum concentrations in NASH compared with NAFLD. A recent studynoted decreased levels of LPC and increased bile acids associated withNASH were not demonstrated in mere steatosis (NAFLD). Differencesbetween NAFLD and NASH appear to be due to the inflammatory component ofNASH; this inflammatory component is absent in fatty liver (NAFLD).

A similar study found that Lysophosphatidylcholine acyltransferases(LPCAT), which convert LPC to PC were up-regulated in NASH with a200%-400% increase in hepatic LPCAT1, LPCAT2, and LPCAT3 mRNAs. Amechanism proposed by Gonzalez et al suggested that hepatic inflammationcausing activation of TNFα and TGFb1 in turn cause increased hepaticLPCAT activity resulting in lower serum LPC levels.

Cholesterol and Bile Acid Homeostasis

As noted above, the inflammatory component of NASH may be linked tofatty acids transported to the liver from visceral adipose.NASH-associated inflammation is also linked to bile acid disruption inthe liver. Gonzalez et al suggested “. . . the decline in serum LPC andrise in serum bile acids are a signature of the inflammatory componentof NASH, rather than the steatotic component.” Furthermore, theysuggested that hepatic inflammation involving TNFα and TGFb1 activationin the liver causes disruption to bile acids, documenting that theenzymes that uptake the bile salts into the hepatocytes were highlydown-regulated, and the transporters that export bile acids from theliver were highly up-regulated in NASH.

1c. Alcoholic Liver Disease (ALD)—PC, Cholesterol and Bile AcidHomeostasis

PC Homeostasis

ALD has long been implicated in disrupted PC metabolism. A study oncirrhotic patients reported that whether cirrhosis was due to alcohol orHBV, there was a decrease in serum LPC of cirrhotic patients compared tohealthy volunteers. Animal studies have shown that alcohol exposure mayproduce pathologies that correlate with NASH. A study on alcoholicmicropigs initially showed increased hepatic triglycerides, and thenafter six months demonstrated inflammation, steatosis and fibrosis. Theauthors concluded that increased lipid synthesis and reduced LPCsynthesis and export were responsible for the accumulation of hepatictriglycerides in ALD. In another study on athymic nude mice gavaged with5% to 40% ethanol solutions, the findings showed the mice developed amild hepatic hemorrhage and serum PC was elevated. There was a decreasein saturated and monounsaturated LPC, but polyunsaturated LPC waselevated.

Cholesterol and Bile Acid Homeostasis

Metabolomic ALD studies show that ALD-induced cirrhosis is associatedwith higher bile acids and lower LPCs (92,94) in a manner almostidentical with non-alcoholic and HBV-induced cirrhosis.

1d. HBV and HCV—PC, Cholesterol and Bile Acid Homeostasis

PC homeostasis

PC biosynthesis is known to be disrupted in both HCV and HBV. AMetabolomic study in China studied HBV patients with deteriorating liverfunction demonstrated disrupted PC levels. The study describes decreasedPC species combined with an elevation of toxic bile acids,glycochenodeoxycholic acid (GCDCA). Another Chinese study reportedsimilar results when examining the progression of chronic HBV tocirrhosis. The same phenomena are also seen in NALFD/NASH, cirrhosis andHCC. An animal study indicated that HCV alters many pathways in theliver with significant changes in LPCs and bile acids, as well ascarnitine esters, fatty acids, and LPEs.

A study conducted by Metabolon Inc., in association with the UniversityCollege Dublin showed the global effects on the hepatic metabolome ofHCV infection. Comparative analysis of 250 metabolites of normalhepatocytes was compared to the same panel of metabolites fromHCV-infected hepatocytes. The study demonstrated specific changes inmetabolism in HCV-infected hepatocytes at 24, 48 and 72 hours,disrupting many different metabolic pathways as a result of HCVinfection, most notably fatty acid, phospholipid, GSH, amino acid,nucleotide and methylthioadenosine metabolism. The study showed alteredflux through both PC biosynthetic pathways (PEMT and CDP-choline), aswell as alteration to all the other CMP-associated pathways.

Cholesterol and Bile Acid Homeostasis

HCV disrupts many aspects of lipid metabolism. Lipids are necessary forHCV viral assembly and secretion. HCV replication modulates host celllipid metabolism dramatically to enhance its replication. The HCV lifecycle requires numerous lipids, which have been shown to be essentialmodulators of the HCV viral lifecycle.

HCV disrupts cholesterol metabolism by causing proteolytic cleavage ofsterol regulatory element binding proteins (SREBPs), thereby inducingsteatosis. Bile acids are derived from cholesterol and HCV disrupts bileacid metabolism as well as cholesterol metabolism.

Apolipoprotein and VLDL synthesis is altered in HCV and HCV-infectedhepatocytes showed increased production of cholesterol andsphingolipids, both of which HCV utilizes to aid in virion maturationand infectivity. Cholesterol-depleted or sphingomyelin-hydrolysed virushad a negative impact on infectivity. Metabolomic analysis ofHCV-infected hepatocytes showed a significant increase in cholesteroland various sphingoid bases.

An animal study was performed in HCV-infected tree shrews, which showedincreased bile acids and decreased PC availability; both are hallmarksof CLD-induced CPM alterations. Further, it has been established thatHBV, HCV, and NASH all trigger similar metabolomics alterationsinvolving hepatic inflammation-mediated changes to bile acid metabolism.Serum bile acids have been found to be higher in patients with severefibrosis as compared to patients with moderate fibrosis.

A Metabolomic study on HCV-infected human hepatocytes showed that HCVimpaired VLDL production and secretion, which in turn may add to theaccumulation of hepatic triglycerides due to decreased VLDL packaging oftriglycerides for hepatic export.

A recent HBV metabolomic study noted a decline in serum PC species andalso elevated GCDCA levels, one of the main toxic components of bileacids, in HBV-infected patients. Serum bile acids have also been foundto be elevated in HCV patients. As mentioned previously, Mitofusin2(Mfn2) gene expression regulates mitochondrial morphology and signaling.A recent study showed that one of the signature toxic components of bileacids, GCDCA, decreases the expression of Mfn2. Further, stimulation inthe lab of overexpression of Mfn2 “. . . effectively attenuatedmitochondrial fragmentation and reversed the mitochondrial damageobserved in GCDCA-treated . . . ” hepatocytes.

This line of research demonstrates how disruption of PC metabolismaffects other CMP-associated pathways in various ways—increased SAMeflux into the up-regulated PC pathway in the liver has a negative effecton flux of SAMe into the GSH pathway. Decreased GSH contributes to thedisruption of redox homeostasis and the creation of extreme oxidativestress in patients with CLD. In a vicious circle, extreme oxidativestress then further oxidizes existing GSH stores, causing increased GSHcycling. Similarly, decreased PC affects choline metabolism, which inturn controls cholesterol metabolism and bile acid homeostasis. Anincrease in bile acids creates an increase in GDCDA from toxic bileacids. As mentioned above, GDCDA exacerbates extreme oxidative stress bynegatively affecting Mfn2 gene expression. Therefore, disruption of PCavailability affects the GSH pathway, and all linked up-regulated CMPpathways contribute to extreme oxidative stress, which is the hallmarkof CLD.

2. CMP is Associated with Increased Storage of CytosolicTriacylglycerides (TGL) in Fatty Liver and Altered α-Oxidation of FattyAcids (FA)

2a. Non-Alcoholic Fatty Liver Disease (NAFLD)—Triacylglycerides (TGL)and Fatty Acids (FA) Accumulation in the Liver

Visceral adipose supplies fatty acids to the liver for β-oxidation. PCcatabolism is also considered a source of fatty acids for hepaticβ-oxidation. Studies indicate that increased levels of hepatictriacylglycerols are also seen in CMP-associated CLD and that thosetriacylglycerols may be exported from adipose tissue.

Studies have also indicated that visceral adipose, known for beinghighly inflammatory, may be responsible for exporting the inflammatorycomponent which distinguishes NAFLD from the inflammatory state of NASH.

In fact, both visceral adipose and up-regulated PC metabolism areconsidered sources of hepatic fatty acids associated with increasedhepatic triacylglycerols. Increased catabolism of PC by phospholipasesA1 and A2 in the liver release free fatty acids. These free fatty acidsrequire β-oxidation in the mitochondria of hepatocytes. Un-oxidizedfatty acids are repackaged as hepatic triacylglycerols, creating NAFLDand setting the stage for NASH.

As noted in section 1b, NAFLD is considered to be the physicalmanifestation of metabolic syndrome in the liver. Metabolomic studieshave found increased lipid species triacylglycerides and diacylglceridesin both liver and blood samples of NAFLD patients.

2b. Non-Alcoholic Steatohepatitis (NASH)—Triacylglycerides (TGL) andFatty Acids (FA) Accumulation in the Liver

NAFLD and NASH are both characterized by changes in the cellular lipidprofile. NASH is NAFLD with a major inflammatory component. For yearsresearchers have been searching for the cause of NASH-associated hepaticinflammation, and new studies theorize that adipose tissue, which isintrinsically pro-inflammatory, may be the origin of hepaticinflammation. As mentioned previously, Lipidomic studies show similarsignificant lipid metabolic disruption in both NASH and NAFLD with onlysmall differences in three PC species to distinguish the two. In NASH aswell as NAFLD, triacylglycerols and several fatty acids were shown to beelevated in plasma, while various other fatty acids showed decreasedlevels in plasma. Other studies also showed that NASH patients withcirrhosis show reduced cellular carnitine levels, which may negativelyaffect β-oxidation of fatty acids in the mitochondria.

2c. Alcoholic Liver Disease (ALD)—Triacylglycerides (TGL) and FattyAcids (FA) Accumulation in the Liver

Early on, Leiber found a correlation with ethanol intake and theaccumulation of hepatic fatty acids and triglycerides. Leiber alsoshowed that ethanol has extreme effects on lipid peroxidation.Metabolomic studies suggest that hepatic fatty acids andtriacylglycerides increase and plasma fatty acids and PC speciesdecrease in ALD.

2d. HBV and HCV—Triacylglycerides (TGL) and Fatty Acids (FA)Accumulation in the Liver

In Section 1 we showed that CLD-associated CMP exhibits up-regulatedcholesterol utilization, which supplies the liver with fatty acids fromPC catabolism. These PC-derived hepatic fatty acids require β-oxidationor they will be re-packaged as hepatic triacylglycerols, contributing tofatty liver. We have shown in Section 1d above that both HCV and HBVinfections affect PC metabolism and increase hepatic fatty acids andtriacylglycerides.

We also discussed that visceral adipose is also a source for bothhepatic fatty acids and hepatic triacylglycerols. The HCV-infected treeshrew study (Tupaia belangeri chinensis) suggested that HCV causesalterations in carnitine esters and fatty acids.

HCV patients have been shown to have low levels of carnitine species,which impairs β-oxidation of fatty acids. An extensive metabolomicsstudy on HCV-infected hepatocytes showed that HCV infection causes anincrease in lipid content within hepatocytes, or liver steatosis.Steatosis is the accumulation of intracellular fatty acids in the liver,and it has been associated with HCV infection, increased oxidativestress and progression to liver cirrhosis. HCV causes proteolyticcleavage of sterol regulatory element binding proteins (SREBPs), therebyincreasing cholesterol and inducing steatosis.

Further, fatty acid oxidation is disrupted during HCV infection and itis associated with HCV-induced metabolic L-Carnitine deficiency. Ametabolomic analysis of HCV-infected hepatocytes showed that HCVdisrupts fatty acid β-oxidation and fatty acid transport to themitochondria. The study also showed that HCV-infected hepatocytes havean increase in fatty acid concentration and a decrease in mediators offatty acid transport Acyl-carnitine (necessary for fatty acid transportto the mitochondria from the cytosol) and proper mitochondrial functionhave been shown to be depleted in HCV. Further, the metabolomic studyshowed a significant decrease in coenzyme A (CoA), pantothenic acid,acetylcarnitine and a number of carnitine derivatives at 48 and 72 hourspost-infection, indicating that fatty acid transport to the mitochondriamay also be disrupted.

3. CMP is Associated with a Shift from Aerobic Respiration to AnaerobicGlycolysis (Warburg Shift)

In the Introduction, we described the new distinctive CMP as itprogresses through the different phases of CLD. One of theCMP-associated changes we noted was the “Warburg shift”, which involvesa shift in metabolism from aerobic respiration to anaerobic glycolysis.This shift to anaerobic glycolysis has usually been associated with HCC,but recent metabolomic studies have shown that the shift happens earlyin all CLD.

As stated previously, the shift from aerobic oxidative phosphorylationto cytosolic anaerobic glycolysis is a hallmark of CLD-associated CMP.The Warburg shift is associated with alterations of metabolic pathwaysthat are linked to both cell proliferation and nutrient acquisition,while the shift to anaerobic glycolysis comes at the expense of ATPproduction.

3a, 3b. NAFLD and NASH—Warburg Shift

Insulin resistance is associated with NAFLD/NASH. In mice, insulin isreported to activate pyruvate kinase M2, which is the enzyme thattriggers cytosolic glycolysis involved in the Warburg shift. The shiftfrom aerobic respiration to anaerobic glycolysis results in theproduction of lactate and alanine from pyruvate. The Warburg shiftoccurs as early as Phase 1 in NAFLD/NASH progression.

3c. ALD—Warburg Shift

ALD progresses towards a shift in aerobic respiration to anaerobicglycolysis along the parallel path of fibrosis/cirrhosis, CMP expressionand HCC development

3d. HBV and HCV—Warburg Shift

Metabolomic studies have documented the Warburg shift occurs in alltypes of CLD, including HBV and HCV as early as phase one, long beforethe appearance of HCC.

Furthermore, HCV has been found to shift metabolism from aerobicrespiration towards the pentose phosphate pathway. The pentose phosphatepathway is a parallel pathway with anaerobic glycolysis. In other words,the pentose phosphate pathway creates NADPH and five-carbon sugars,which are then oxidized by glycolysis in HCV patients.

4. CMP is Associated with GSH Depletion, increased Consumption ofThiol-containing Metabolites, Extreme Oxidative Stress and AlteredOne-Carbon Metabolism

CLD of all etiologies are associated with CMP-induced hepatic extremeoxidative stress. Glutathione is the master controller of cellular redoxstatus, maintaining and regulating the redox status of cellularenzymatic and non-enzymatic (small-molecule) antioxidants.CLD-associated depletion of GSH may cause depletion of all linkedcellular antioxidants that are normally maintained and regulated by GSH.

4a, 4b. NAFLD and NASH—Altered Homocysteine/Glutathione/One-CarbonMetabolism

Metabolomic analyses on a variety of types of NAFLD samples have beenperformed on animal models, living human subjects and human tissuesamples. One common finding is that cysteine-glutathione disulfide andboth oxidized and reduced glutathione were depressed in the liver andserum/plasma, and this condition is associated with extreme oxidativestress in patients with NAFLD/NASH.

The progression of steatosis (NAFLD) to steatohepatitis (NASH) involvesthe sensitization of hepatocytes through oxidative stress tocytokine-induced apoptosis and the importation of inflammatory fattyacids from adipose tissue. Depletion of mitochondrial glutathione (mGSH)is associated with cholesterol infiltration of mitochondrial membranes,which lowers transmembrane potential, and which in turn inhibits thetransport of cytosolic GSH into the mitochondrial membrane. Glutathionedepletion has been found to be involved with hepatic stellate cellactivation.

One of the most common research models for inducing steatosis is themethionine and choline deficient diet (MCD). MCD is associated withsteatosis, mitochondrial dysfunction, hepatocellular injury, oxidativestress, inflammation, and fibrosis. One study analyzed the contributionsof both the methionine deficient diet (MD) and the choline deficientdiet (CD) to total MCD pathogenic effect. They found that MD reproducedmost of the deleterious effects of the total MCD, while CD caused mainlysteatosis, with a rise in hepatic FA and TGL accumulations, but withoutmuch of the other deleterious effects associated with MCD. The studyfound that S-adenosylmethionine (SAMe) and glutathione (GSH) depletionin the mitochondria precede the observed effects due to decreasedmitochondrial membrane fluidity associated with a lowerphosphatidylcholine/phosphatidylethanolamine ratio.

GSH or SAMe therapy restored GSH in the mitochondria and amelioratedhepatocellular injury in mice fed either a MCD or MD diet

4c. ALD—Altered Homocysteine/Glutathione/One-Carbon Metabolism

Alcohol toxicity has also long been linked to folate/homocysteine orone-carbon metabolism disruption. Early research established significantethanol-associated glutathione depletion and oxidative stress, alteredmethionine metabolism, altered folate/homocysteine/one-carbonmetabolism, malnutrition, and increased Kupffer cell activation. Alcoholinduced oxidative stress and inflammation has been shown to exacerbatethe progression of the disease.

Changes in gene expression can be accomplished through alterations inDNA coding sequence. Changes in gene expression and phenotype may alsobe caused by other mechanisms. Epigenetics is the study of theseheritable changes. DNA can be modified epigenetically by DNAmethylation, histone modifications, and RNA-based mechanisms. Recentstudies have focused on epigenetic features, transcriptional factors andsignaling pathways associated with chronic ALD. These new studiesprovide greater nuanced perspectives of ALD/CLD disease pathology.Consumption of ethanol causes epigenetic changes (CMP) that contributeto ALD. In an extensive study, ethanol affected metabolism of methionineand thereby DNA methylation. ALD is associated with GSH depletion in themitochondria. Studies show that ALD-induced mitochondrial GSH depletionis associated with cholesterol-enrichment of the mitochondrial membrane,which leads to impairment of GSH transport of cytosolic GSH into themitochondria in both ALD and NAFLD/NASH.

4d. HBV and HCV—Altered Homocysteine/Glutathione/One-Carbon Metabolism

Both HCV and HBV induce hepatic oxidative stress. The mechanisms forincreasing oxidative stress in both pathologies are well known. Thesemechanisms involve epigenetic changes caused by viral proteinsinteracting with both mitochondria and endoplasmic reticulum (ER) toincrease mitochondrial reactive oxygen species (ROS) generation. HCVcore expression inhibits electron transport at Complex I, and increaseComplex I ROS production. They also induce depletion of mitochondrialGSH, increase mitochondrial membrane permeability and impair variousantioxidant defense mechanisms.

A metabolomic study on HCV-infected hepatocytes showed changes to theGSH pathway at 24, 48 and 72 hours, demonstrating that HCV caused six orseven specific epigenetic changes to the metabolome. These findings areconsistent if not identical with the phenotypic expression of the CMPseen in all forms of CLD.

HCV-mediated mitochondrial disruption is a causative factor in GSHdepletion and the creation of extreme oxidative stress. HCV has beenshown to create replication sites in the mitochondrial membrane thatdamages mitochondrial form and function. HCV has also been shown todisrupt the GSH metabolic pathway, causing greater utilization of GSHand disrupted biosynthesis. HCV-impaired GSH biosynthesis contributes toa large increase in ROS generation that further contributes tomitochondrial GSH depletion.

5. CLD/CMP-Associated Extreme Oxidative Stress and Fibrogenesis

CLD causes extreme oxidative stress as a result of CMP-associatedmetabolic disruptions and unfulfilled distinctive nutritionalrequirements. NASH, ALD and HBV all have been associated with extremeoxidative stress. CLD-induced metabolic changes increase ROS and NOSlevels, which disrupt redox homeostasis and create extreme oxidativestress. Depletion of GSH results in the inability to counteractoxidative-mediated insults to cellular systems, resulting inirreversible cellular degeneration and cell death. GSH depletion andCLD-associated oxidative stress may damage mitochondria form andfunction. Furthermore, non-enzymatic small molecule antioxidants andantioxidant minerals are both depleted as a result of CLD-inducedoxidative stress, GSH depletion and cirrhosis.

GSH is the main regulator of cellular redox homeostasis and redox signaltransduction. Under normal metabolism, cellular redox status ismaintained and regulated by the glutathione redox couple GSH:GSSG (GSSGis an oxidized form of GSH), along with the NADPH/NADP+ andTrx-SH/Trx-SS redox couples. Redox balance is involved in cellularsignal transduction, and small changes in the GSH:GSSG ratio areinvolved in the fine-tuning of signal transduction in physiologicalevents such as cell cycle regulation and other processes.

The interaction of GSH and the free-thiol in cysteine residues forms amixed disulfide that is reversibly formed throughprotein-S-glutathionylation to protect proteins from irreversibleoxidative stress. Protein-S-glutathionylation is an important mechanismfor post-translational regulation of a large list of regulatory,structural and metabolic proteins that play roles in cell signaling andmetabolic pathways. Protein-S-glutathionylation production requires areactive cysteinyl residue, present at physiological pH in the thiolateform. Under oxidative stress, these cysteinyl residues may be oxidizedand react with GSH leading to a glutathionylated-cysteine derivative.Both GSSG and S-glutathionylated proteins may be catalytically reducedback to GSH, while they both may also be reduced back to GSHnon-enzymatically.

Protein-S-glutathionylation is an important mechanism forpost-translational regulation. Regulatory, structural and metabolicproteins that react with GSH to form S-glutathionylated proteins areinvolved in cell signaling and the regulation of cellular metabolicpathways.

Disruption of GSH-controlled cellular redox homeostasis increasesoxidative stress. Disruption of GSH homeostasis has been implicated inthe pathogenesis and progression of many human diseases. Decreased GSHlevels contributing to oxidative stress have been associated with aging,neurodegeneration, inflammation, and infections.

Besides countering ROS-associated oxidative stress, GSH is alsocritically involved in mediating the susceptibility of nitric oxide (NO)and NO derivatives in the body. GSH is involved in countering ReactiveNitrogen Species (RNS) associated oxidative stress and counteractingRNS-mediated damage. CLD increases ROS and NO levels, which disruptredox homeostasis and create extreme oxidative stress. As mentionedabove, depletion of GSH results in the inability to counteract oxidativestress and NO-mediated insults to hepatic cellular systems, resulting inirreversible cellular degeneration and cell death.

Extreme hepatic oxidative stress and oxidative stress-mediated hepaticinflammation trigger the separate process of liver fibrogenesis. Hepaticfibrogenesis involves a process whereby CMP-associated oxidative stresstriggers the conversion of normally quiescent hepatic stellate cellsinto active collagen-secreting myofibroblasts.

Part B: The Medical Food Contemplated herein Fulfills the newDistinctive Nutritional Requirements for Patients with CLD

In Part A we established that all forms of CLD create distinctiveepigenetic changes in the phenotype and alterations of metabolism inpatients with CLD. We also established that these changes are similar,and in most cases identical in all forms of CLD, regardless of thedisease etiology. Extensive metabolomic studies on all forms ofhepatobiliary diseases have collectively labeled these new distinctivemetabolic/phenotypic changes as the new Core Metabolic Phenotype (CMP)for CLD patients.

As noted previously, these CMP phenotypic changes are very similar inall CLD, whether they involve epigenetic changes and/or metabolicalterations associated with chronic ALD, NAFLD, NASH or oncogenicviruses like HCV or HBV. These CMP-associated changes result in theup-regulation of specific biosynthetic metabolic pathways, whichincreases the metabolic demand for metabolites and end-products involvedin those biosynthetic pathways.

A person's complete nutritional requirements are defined as the sum of aperson's combined metabolic demands. Therefore the new distinctivenutritional requirements of CLD patients must factor in increased demandfor metabolites and end-products of up-regulated biosynthetic pathwaysassociated with CLD.

A medical food for CLD patients must strategically identify, coordinateand supply the proper amount and balance of metabolites necessary tofulfill the new distinctive nutritional requirements of CLD patients.Disrupted CMP-associated metabolic pathways are listed below:

-   -   Dis-regulation of phospholipid metabolism and membrane        phospholipid reallocation,    -   Dis-regulation of cholesterol and bile acid homeostasis,    -   Increased storage of cytosolic triglycerides and fatty acids        accompanied with altered β-oxidation of fatty acids,    -   A shift from aerobic respiration to anaerobic glycolysis,    -   Increased utilization of thiol-containing metabolites including        increased glutathione/cysteine cycling, increased        SAM/SAH/methionine cycling and increased one-carbon methylation        metabolism

The CMP-related metabolic alterations listed above involve up-regulationand increased cycling of metabolites involved in the followingup-regulated biosynthetic pathways:

-   -   * The GSH pathway as well as other GSH-linked antioxidant        systems, including cellular small molecule non-enzymatic        antioxidants and mitochondrial metabolites    -   * Both phosphatidylcholine (PC) biosynthetic pathways (PEMT and        CDP-choline) and mitochondrial metabolites associated with        mitochondrial membrane form, function and transmembrane        potential    -   * The SAM/SAH/methionine cycle with associated one-carbon        methylation metabolism    -   * Amino acid metabolism

There is ample evidence that depletion of biosynthetic metabolites inone CLD-altered pathway may exacerbate other existing CLD-alteredpathways. This is due to the fact that CLD/CMP-altered biosyntheticpathways are interlinked and most of these up-regulated pathways drawdirectly on SAMe stores. These interlinked pathways include the GSH andPC pathways as well as the SAM/SAH/methionine cycle and one carbonmetabolism. Nutritional depletion of key nutrient metabolites in theseinterlinked metabolic pathways may have a synergistic effect onpromotion of extreme oxidative stress, which is the hallmark of CLD.

As noted in Section A, extreme hepatic oxidative stress and oxidativestress-mediated inflammation triggers the process of hepaticfibrogenesis. Fibrogenesis involves the conversion of hepatic stellatecells into myofibroblasts. Myofibroblasts secrete collagen into theextracellular spaces of the liver, resulting in fibrosis and cirrhosis.

While liver fibrosis was once considered irreversible, modern studieshave demonstrated that once the trigger to fibrogenesis (extremeoxidative stress) is switched off, myofibroblasts apoptosis may occurwhile hepatic stellate cells remain quiescent and the process ofcollagen resorption may then proceed at a fairly constant rate. Theprocess of collagen resorption is well established and has beendemonstrated in numerous animal and human CLD studies.

SAMe is the most ready methyl donor of all the one-carbon methylationdonors, so it is no surprise that all CMP up-regulated biosyntheticpathways similarly draw on SAMe. SAMe is also involved in thetranssulfuration pathway in GSH synthesis. Further, there is a largebody of existing current and historical studies involving exactly thesesame CMP metabolic pathway alterations and the interlinked effects thatone altered pathway may have on other CMP pathways. Dr. C. S. Leiber wasa pioneer in this area and performed a series of seminal studies on ALDand liver cirrhosis.

CMP alterations establish a vicious positive feedback loop regardingCLD-induced oxidative stress: CMP-altered metabolic pathways createextreme oxidative stress, and extreme oxidative stress negativelyaffects the altered CMP metabolic pathways. Extreme oxidative stresscauses increased oxidation of: PC to oxidized PC; methionine tomethionine sulfate; homocysteine to homocystine; cysteine to cystine;GSH to GSSG, GCDCA in bile acid, and proteins to PrS-SC, PrS-SG, andPrS-SCG. These oxidized metabolites require reduction to reactivatetheir healthy roles in metabolism. Accumulation of oxidized forms ofthese metabolites may have negative implications for health. Forinstance, oxidized PC may cause apoptosis in macrophages and affect cellviability ( ).

A recent review of GSH studies concluded that, “Conditions characterizedby increased ROS levels may require not only enhanced GSH action tomaintain redox status, but also augmented energy supply and precursorsto replace/enhance GSH content and/or transport it to the places whereit is needed”. Further, extreme oxidative stress may decrease SAMeavailability, which contributes to DNA hypomethylation and oxidation.DNA hypomethylation may induce and/or exacerbate further alterations toCMP gene expression. Extreme oxidative stress has been identified as atrigger to hepatic stellate cell conversion in the process of hepaticfibrogenesis.

PC supplementation has also been studied in CLD, as has SAMesupplementation. While animal studies have shown promise, human studiestargeting individual metabolites have been inadequately controlled andhave shown mainly inconclusive results. There have been calls for humanclinical trials involving combinations of these CLD up-regulatedmetabolites.

The medical food clinical trials will demonstrate that its treatmentserves to decrease oxidative stress and/or re-establish redoxhomeostasis in study patients. This in turn will down-regulatefibrogenic activity and slow down or stop the progression of fibrosiswhile allowing the reversal of fibrosis to occur. This reversal of liverfibrosis is a primary and unexpected advantageous result of the presentinvention. Reports of reversal of fibrosis in ALD patients who have quitdrinking vary and have been inconsistent, while there has been a dearthof studies regarding this topic. Therefore, clinical trials of thepresent invention will determine if and/or when reversal of fibrosis ispossible in non-drinking, compliant patients with ALD. If the initialclinical trial finds that reversal of fibrosis is possible in ALDpatients, future studies on non-drinking, compliant patients with ALDmay be necessary to determine whether reversal of fibrosis may beinduced or accelerated by fulfilling the distinct nutritionalrequirements of patients with ALD and stabilizing their alteredmetabolic pathways. Meanwhile, past studies have shown reversal offibrosis after the underlying etiology is eliminated in HCV, HBV, NASHautoimmune hepatitis, and secondary biliary fibrosis. The medical foodclinical trials will investigate whether the fibrogenic activityassociated with CLD may be down-regulated by fulfilling the distinctivenutritional requirements of CLD patients, and furthermore, whetherreversal of fibrosis may be accomplished even in the presence ofcontinuing CLD in the cases of HCV, HBV and NAFLD/NASH patients or inthe chronically altered phenotype of non-drinking, compliant ALDpatients.

The medical food clinical trials will monitor changes in liver fibrosisstaging in CLD patients over time. Any positive improvement in fibrosisstaging may be due to down-regulation of the fibrogenic activationcaused by fulfillment of the distinctive nutritional requirements of theCLD patients. Conversely, any additional progression of fibrosis may beassociated with the direct action of CLD rather than unfulfillednutritional requirements of the CLD patients.

Previous Studies

Over the last several decades Dr. C S Lieber published over 80 studieson ALD, cirrhosis, SAMe and PC. Dr. Leiber was responsible for manyimportant discoveries including the microsomal ethanol oxidizing system(MEOS) involving cytochrome P4502E1. Dr. Leiber performed many importantanimal studies involving PC and SAMe administration in ALD. Early on,Dr. Leiber noted that PC supplementation decreased oxidative stress,correctly observing that supplying the biosynthetic end-product of thePC biosynthetic pathway decreases draw on SAMe, which is thenre-directed to GSH biosynthesis. Dr. Lieber observed that the resultingup-regulation in GSH production was responsible for the observeddecrease in oxidative stress.

Dr. Leiber went on to study PC and SAMe administration in rats andbaboons fed ethanol, noting that PC administration attenuated CC14 andethanol-induced liver injury, while SAMe restored hepatic GSH levels andhad a positive effect on mitochondrial lesions and leakage. In 2002, Dr.Leiber pointed to promising animal studies and called for human studiesinvolving administration of SAMe to patients with ALD, noting that “. .. therapeutic administration of SAMe should be the subject of acomprehensive clinical trial to assess its capacity to attenuate earlystages of alcoholic liver injury in human beings.”

Numerous other studies, for instance, a 1989 Scandinavian human studyreached the same conclusions, seeing a “significant increase” in levelsof hepatic glutathione, in patients with both alcoholic andnon-alcoholic liver diseases—“SAMe may therefore exert an important rolein reversing hepatic glutathione depletion in patients with liverdisease.”

Dr. Leiber pointed out that the immediate metabolic precursor of SAMe ismethionine, but methionine must be enzymatically activated to SAMe, andthis enzyme is impaired in ALD. Therefore, he warned againstadministering methionine instead of SAMe due to this enzymaticinhibition,

-   -   “The precursor of SAMe is methionine, one of the essential amino        acids, which is activated by SAMe-synthetase (EC 2.5.1.6).        Unfortunately, the activity of this enzyme is significantly        decreased as a consequence of liver disease. Because of        decreased utilization, methionine accumulates and,        simultaneously, there is a decrease in SAMe that acquires the        status of an essential nutrient and therefore must be provided        exogenously as a super nutrient to compensate for its        deficiency.”

A 2011 review described various studies regarding the efficacy oftreating patients with ALD metabolic disorders with SAMe. The authorsconclude that because recent SAMe studies are inconclusive orcontradictory, the one-carbon methyl donors associated withhomocysteine/methionine conversion should be included in future SAMe ALDstudies,

-   -   “The doors have now been opened for potentially productive        research into the relationship of epigenetic changes in        SAM-regulated gene methylation to all pathways of liver injury        in ALD. Furthermore, the inconclusive results of trials in SAM        treatment of ALD suggest that provision of other nutritional        factors involved in SAM metabolism, such as vitamin B-6, should        be included with SAM in larger and more prolonged clinical        trials.”

A symposium titled, “Role of S-Adenosyl-L-Methionine (SAMe) in theTreatment of Alcoholic Liver Disease” was sponsored by The NationalInstitute on Alcohol Abuse and Alcoholism and the Office of DietarySupplements, National Institutes of Health in Bethesda, Md., September2001, also discussed SAMe with a potential role in ALD:

-   -   “The presentations of this symposium support the suggestion that        SAMe may have potential to treat ALD by (1) acting as a        precursor of antioxidant glutathione, (2) repairing        mitochondrial glutathione transport system, (3) attenuating        toxic effects of proinflammatory cytokines, and (4) increasing        DNA methylation.”

Dr. Leiber studied SAMe depletion in early ALD and noted that decreasedSAMe levels occur even before SAMe-synthetase is inhibited. Dr. Leiberattributed SAMe depletion to extreme oxidative stress associated withthe metabolism of alcohol, which rapidly consumes GSH. Because SAMe isone of the rate-limiting steps in GSH biosynthesis, exogenous SAMeadministration was an object of many studies by Dr. Leiber on patientswith ALD, and his conclusions all favored human clinical trialsinvolving SAMe and PC administration in patients with CLD.

However, several recent SAMe studies on CLD have shown inconclusive orinconsistent results. One recent study showed that oral SAMeadministration has low bioavailability, and the authors recommendesterifying the molecule to form a “more lipid-soluble prodrug”.Therefore, low oral SAMe bioavailability may be a significantcontributing factor to the inconsistent results seen in various recentSAMe studies. Further, SAMe is contraindicated for patients with bipolardisorders and Parkinson's disease and it is associated with serotoninmetabolism, so use of serotonin-related drugs are contraindicated forSAMe use as well.

For the reasons listed above, the present invention does not includeSAMe as a required metabolite in its formula. The medical foodcompensates for this omission in three ways:

First, it has been proposed above that SAMe contributes four majorbenefits in CLD; further, increased GSH production by SAMe is generallyregarded as the most significant benefit. Significantly, there are tworate-limiting metabolites in the GSH biosynthetic pathway:S-adenosylmethionine (SAMe) and N-acetylcysteine (NAC). In other words,administration of either SAMe or NAC will promote GSH production. Themedical food of the present invention chooses to fulfill the distinctivemetabolic requirements of the CMP-associated up-regulated GSH pathway byproviding NAC as the rate-limiting metabolite instead of SAMe tostabilize the GSH pathway and to increase GSH synthesis. Consumption ofa cysteine-supplement such as L-cysteine or NAC safely and effectivelyincreases GSH production without affecting the SAM/SAH/methioninepathway; thereby fulfilling CMP-increased metabolic demand for GSHmetabolites while simultaneously saving SAMe for other uses includingproper DNA methylation. While NAC is used herein as an example, itshould be understood that other cysteine-providing ingredients such asL-cysteine may be used herein as well. Importantly, proper GSHbioavailability will work to reduce CMP-associated oxidative stress andCLD metabolic perturbations associated with extreme oxidative stress.

Second, just as Dr. Leiber noted early on, providing PC decreasesoxidative stress by reducing the flux of SAMe into the highlyup-regulated PC pathways in CLD. Therefore, PC administration resultedin more SAMe availability for GSH production. In the same way,administration of NAC also increases SAMe availability, because NACadministration reduces the draw of SAMe into the GSH pathway. The resultof fulfilling the increased metabolic demand for metabolites in both ofthese up-regulated CMP-associated pathways is that SAMe is lessutilized, saving SAMe for other metabolic functions, including DNAmethylation.

Third, supplementing the one-carbon metabolite betaine correctsSAMe-synthetase deficiency and effectively restores methionineconversion to SAMe, as Dr. Leiber pointed out that SAMe-synthetase isinhibited in ALD. In fact, administration of one-carbon metabolites hasbeen shown in many CLD studies to restore proper SAM/SAH/methioninecycling, including homocysteine to methionine cycling, and methionine toSAMe cycling. Many researchers have concluded that future glutathionestudies must necessarily include one-carbon metabolites as well asmetabolites of the SAM/homocysteine cycle.

Most of the nutritional studies to date have focused on singlemetabolites or single metabolic pathways in various conditions of CLD.However, the interlinked nature of these CMP-altered metabolic pathwaysbeg for a comprehensive and systematic approach to fulfill thedistinctive nutritional requirements of CLD patients. For instance, manyresearchers have stated the need to add the one-carbon methyl donors toany future CLD studies involving SAMe. As noted previously, manyresearchers have advocated a multi-ingredient approach to future humanCLD clinical trials due to the interlinked nature of the CLD affectedmetabolic pathways. The medical food supplies the one-carbonmetabolites, as well as the precursor metabolites and the end-productmetabolites to all CLD-affected biosynthetic pathways. The medical foodphase II human clinical trials will administer CMP-associatedmetabolites in a comprehensive and systematic approach to fulfillup-regulated CMP metabolic demand with proper levels of requiredmetabolites. The medical food clinical trials will examine the effectsof this regimen using FibroScan, a non-invasive device, to assess liverstiffness, which correlates well with fibrosis staging in patients withCLD.

The medical food does not treat CLD—it will not cure or treat HCV, HBV,NASH or ALD; rather it satisfies the new distinctive nutritionalrequirements of patients with CLD. Extreme oxidative stress is createdin CLD in part as a result of unfulfilled nutritional requirements ofpatients. Fulfilling the distinctive nutritional requirements of CLDpatients may therefore decrease oxidative stress. If long-termre-establishment of redox homeostasis may be achieved, then improvementin fibrosis staging may be possible. This is because fibrogenesis is nota direct action of CLD. Fibrogenesis is a separate hepatic process thatis triggered by extreme oxidative stress associated with CLD, butfibrosis is not caused by any direct action of CLD. In fact, GSHdepletion and oxidative stress have been implicated in triggeringfibrogenesis through hepatic stellate cell conversion to myofibroblasts.Metabolomic studies have implicated CMP-associated metabolic disruptionsto the creation of CLD-associated extreme oxidative stress. In otherwords, CLD-associated extreme oxidative stress is at least in part theresult of unfulfilled metabolic requirements of patients with CLD,resulting in decreased availability or depletion of necessarymetabolites of the PC, GSH, SAMe, and one-carbon metabolic pathways.Extreme oxidative stress is associated with the trigger to fibrosisgeneration; therefore if redox homeostasis is re-established,down-regulation of the fibrogenic process may be achieved. The medicalfood of the present invention's intended use is to re-establish redoxhomeostasis by fulfilling the metabolic/nutritional demands of the CLDpatients. Studies have shown that once the fibrogenic activity iseliminated, resorption of hepatic collagen may then occur therebydecreasing fibrosis staging.

The Present Medical Food Invention Fulfills the new DistinctiveNutritional Requirements of CLD Patients

Present Invention Supplies Metabolites involved in Glutathione (GSH)Biosynthesis

Glutathione (GSH) in the body is intrinsically involved with cellularredox homeostasis, and therefore GSH homeostasis is important in anydisease that causes increased levels of oxidative stress. As notedpreviously, extreme oxidative stress is a hallmark of CLD and GSH isdepleted due to decreased production and overconsumption. Also, GSHexperiences decreased production in CLD due to CMP disruption of theSAMe cycle and one-carbon metabolism. Therefore, researchers have calledfor studies that investigate the administration of precursor metabolitesof GSH synthesis to patients with diseases associated with metabolicdepletion of GSH, describing it as a step that is important for researchefforts into a variety of chronic diseases.

GSH is essential for cellular redox homeostasis and GSH synthesis istightly regulated in the cytosol. After synthesis, GSH is distributed tointracellular compartments such as the mitochondrial membrane,endoplasmic reticulum and the nucleus. It is also exported toextracellular spaces including the blood and bile for utilization byother tissues. The half-life of GSH is only 2-3 hours. GSH is onlycatabolized in the extracellular space by gamma-glutamyl transferase(GGT). The gamma glutamyl cycle involves the rapid catabolism ofextracellular GSH into constituent peptides, which are then quicklytaken back up by the cells for rapid re-synthesis into GSH. This gammaglutamyl cycle of GSH cellular export/catabolism/re-uptake/re-synthesismay be energy inefficient, but it is a perfect design for arapid-response antioxidant system in response to extreme oxidativechallenges. Intracellular GSH status depends on precursor availability,the rate of GSH oxidation to GSSG, and the capacity to recycle GSSG backto GSH at the expense of NADPH. Under normal physiological conditions,reduced GSH levels are 10 to 100 times greater than oxidized GSH (GSSG)and mixed disulphide (GSSR). The ratio of reduced and oxidized forms ofGSH is important in cell signaling, maintaining redox homeostasis andthe promotion of cellular mechanisms associated with cell proliferation,cell differentiation or apoptosis, while small variations in GSH:GSSGratio tightly regulate redox signaling.

GSH depletion has been associated with inhibition of cytochrome coxidase (CcOX) activity, microtubule network disassembly, and processesassociated with NO toxicity.

In GSH biosynthesis, GSH is produced through the transsulfurationpathway involving SAMe conversion (cycling) to homocysteine, then toNAC. NAC and L-Glutamate are then combined into gamma-glutamylcysteineby the enzyme gamma-glutamyl synthetase—the rate-limiting step in thebiosynthesis of GSH. Glycine is then added to the C terminal of thegamma-glutamylcysteine molecule by the action of the enzyme glutathionesynthetase.

CMP-associated oxidative stress induced by CLD increases GSH activityand consumption, which in turn prompts changes in GSH levels. CLDcreates a distinctive nutritional requirement for increased GSHproduction. As noted above, GSH is depleted in CLD, causing demand forgreater synthesis and flux of SAMe and other GSH metabolites through theGSH pathway. GSH depletion has been shown to be involved in hepaticstellate cell activation in fibrogenesis.

GSH performs a variety of metabolic roles in the body, includingantioxidant functions as a radical scavenger and as a redox signalingmodulator. GSH scavenges free radical ROS and RNS directly andindirectly through enzymatic reactions. GSH also reacts enzymaticallywith hydroperoxides, being a co-substrate for selenium-dependentGlutathione Peroxidase (GPX), which is the body's most importantmechanism for reducing H2O2 and lipid hydroperoxides. GSH may alsoreduce and detoxify ROS-promoted lipid-oxidation products such asmalonyl dialdehyde and 4-hydroxy-2-nonenal, as well as many otherspecies. GSH maintains thiol homeostasis of cysteine residues onproteins. It also conjugates and stores cysteine reserves. Glutathioneis associated with estrogen, leukotriene, and prostaglandin metabolism.GSH also participates in the production of deoxyribonucleotides, in thematuration of iron-sulfur cluster in proteins, and it participates insignal transduction and cellular transcription.

As noted previously, the medical food has chosen to include NAC toincrease GSH production rather than SAMe. Administration of NAC has alsolong been known to safely promote intracellular GSH production, decreaseoxidative stress and has had anti-fibrotic actions in preliminary humanstudies. NAC has also been shown to decrease inflammatory markers anddecrease hepatic fatty acid accumulation in ethanol-fed rats. Theaddition of NAC to corticosteroids has also been shown to decreasehepatorenal syndrome, infection, and short-term mortality in patientswith severe ALD.

The medical food supplies NAC for GSH synthesis, which decreases demandfor SAMe because SAMe is normally converted to NAC for GSH synthesis. Asstated previously, the present invention also provides PC, which alsodecreases SAMe utilization in the PC PEMT biosynthetic pathway in CLD.Administration of NAC and PC, both of which are metabolic end-productsof CMP-disrupted metabolic pathways, conserve SAMe for other purposes,including DNA methylation or some of the many other metabolic functionsof SAMe.

CLD is associated with DNA hypomethylation. DNA hypomethylation resultsin phenotypic and epigenetic alterations. ALD causes alcoholic steatosisand methionine metabolism disruption associated with DNA hypomethylationand altered gene expression. SAMe is one of the main one-carbon methyldonors that methylates DNA. However, SAMe availability is limited byimpaired enzyme activity in the SAM/SAH/methionine cycle in ALDpatients, and decreased SAMe is one of the causes of DNAhypomethylation. The medical food restores SAMe availability byadministering all of the one-carbon methyl donors associated with CMPpathways, as well as by saving SAMe from overutilization in the GSH andPC pathways. In this way, the medical food's one-carbon metabolitessafely restore enzymatic cycling of the homocysteine/SAH/methioninepathway to produce more SAMe for proper DNA methylation.

CLD is also characterized by increased mitochondrial permeabilitytransition, and this is associated with ROS penetration into thecytosol. NAC has been shown to inhibit alterations of mitochondrialpermeability transition. Therefore, metabolic precursors of the GSHbiosynthetic pathway including NAC, glycine, and glutamate are thedistinctive required nutrients for patients with CLD.

The Present Invention Supplies Metabolites involved in otherGSH-Interlinked Antioxidant Systems

New distinctive nutritional requirements created by CLD necessitatesystematic and comprehensive dietary management. A 1997 review regardingviral diseases and their induction of oxidative stress remarked howcomplex and deleterious the pathogenic induction of oxidative stress is,noting that supplying specific antioxidants may solve both short-termand long-term issues seen in patients with HCV.

GSH biosynthesis is intrinsically involved in the regulation and redoxcycling of various antioxidant systems. In particular, CMP up-regulatedthiol-containing antioxidant systems need dietary management to fulfilltheir functions and remain in homeostasis, and the medical food suppliesNAC, ALA and PC to fulfill the distinctive nutritional requirement ofCLD patients for these up-regulated thiols.

The body has two basic antioxidant systems, classified as the enzymaticantioxidant system and the non- enzymatic antioxidant system:

-   -   Enzymatic antioxidants include superoxide dismutase (SOD),        catalase, glutathione peroxidase, thioredoxin and glutaredoxin.    -   Small molecule non-enzymatic antioxidants include lipid soluble        vitamins A, D and E. Vitamins B, C and GSH are water-soluble        antioxidants. GSH is the master controller for proper        cooperative reduction of these linked-chain small molecule        non-enzymatic cellular antioxidants.

Intracellular small molecule antioxidants like vitamins B's, C, D and Eare consumed at an increased rate due to CLD-associated extremeoxidative stress. In addition, trace elements like zinc, selenium, andmanganese are metabolic antioxidant cofactors that also experiencegreater utilization in patients with CLD. Selenium is a cofactor ofglutathione peroxidase and zinc, manganese and copper are cofactors forSOD.

These small molecule antioxidants require reduction after oxidation inorder to re-establish antioxidant function. This is accomplished throughreducing systems such as glutathione/glutathione disulfide,dihydrolipoate/lipoate, or NADPH/NADP+ and NADH/NAD+. These smallmolecule antioxidants also reduce each other in a linked-chainre-charging redox system. For instance, CoQ10 has been shown to enhanceenzymatic NADH- and NADPH-recycling of tocopherols in mitochondriawithout being consumed itself. Vitamin C, ALA and GSH all reduce vitaminE. Vitamin E reduces vitamin A and the carotenoids, while it stabilizesmembranes and protects against lipid peroxidation. ALA and seleniumreduce GSH. GSH reduces cystine, vitamin C and polyphenols. Decreasedlevels of GSH and increased oxidative stress associated with CLD have animpact on the reduction capacity of the small-molecule non-enzymaticantioxidant system, while chronic extreme oxidative stress may impairthe ability of the redox system to maintain cellular redox homeostasis.Due to the interlinked nature of their redox duties, depletion of anyindividual members of this cellular non-enzymatic small-moleculeantioxidant recharging system may result in decreased levels of allmembers of this linked antioxidant system. For these reasons, propercombinations of linked antioxidants are required for full-systemstabilization. GSH also contributes to the redox homeostasis ofmitochondrial antioxidant metabolites, including the B vitamin family,CoQ10 and ALA.

Antioxidant minerals such as zinc, selenium and magnesium are consumedat a higher rate in CLD related to increased oxidative stress. ALD hasalso been shown to impair zinc uptake. Therefore, CLD creates a distinctnutritional requirement for increased intake of these antioxidantmineral metabolites. Zinc, selenium and magnesium are part of therequired nutrients contained in the medical food to meet increaseddemand from CLD-associated elevation of oxidative stress.

The medical food contemplated herein provides the full complement ofinterlinked small-molecule non-enzymatic antioxidants, including theantioxidant minerals zinc, selenium, magnesium, CoQ10, vitamins A, B, C,D, E, and the thiol-based cellular antioxidant ALA in moderate, balancedquantities. The medical food's three times-daily administration scheduleis intended to maximize periods of nutrient delivery in response to theconstant up-regulated metabolic and nutritional demands of patients withCLD.

The medical food nutrients also fulfill a distinctive nutritionalrequirement for B vitamins, which are necessary for proper mitochondrialmetabolism, and which are altered in CLD. Vitamin B deficiencies causemitochondrial dysfunction, and administration of B vitamins mayameliorate symptoms associated with B vitamins deficiencies and preventmitochondrial toxicity ( ).

The medical food fulfills a distinctive nutritional requirement for allthe B vitamins in patients with CLD. This distinctive nutritionalrequirement is due to increased consumption due to CLD-associatedextreme oxidative stress. Niacin (vitamin B3) is a necessarymitochondrial B vitamin and is the precursor of NAD and NADPH. Niacinsupplementation has a positive effect on fatty liver. A study by Li etal found “Chronic EtOH feeding induced significant lipid accumulation inthe liver, which was . . . ameliorated by dietary NA supplementation.Liver total NAD, NAD(+), and NADH levels were remarkably higher in theNA supplemented group than the NA deficient or EtOH alone groups”. NADHreduces oxidized GSH. The medical food supplies thiamine (vitamin B1),another mitochondrial antioxidant B vitamin found decreased in CLD.Thiamine administration reversed many of the detrimental effects ofethanol administration in rats.

Studies showed HCV creates a distinctive nutritional requirement forincreased CoQ10 due to HCV-induced depletion of mitochondrial GSH,increase in ROS production and disruption of the mitochondrial electrontransport chain. CoQ10 is a mitochondrial antioxidant and involved inelectron transport in the mitochondria, Intracellular CoQ10 levelsreflect the functional status of the electron transport complex in themitochondria. CoQ10 reduces GSH without being consumed itself due to itspromotion of enzymatic process.

The medical food formula fulfills the distinctive nutritionalrequirements for vitamin E, selenium, magnesium and zinc, which aredepleted in CLD, in particular HCV infection. Studies demonstratedadministration of these antioxidants alleviated symptoms of oxidativestress in patients with HCV.

The medical food also fulfills a distinctive nutritional requirement formagnesium. Magnesium deficiency is associated with cirrhosis and itplays a significant role in increasing oxidative stress and apoptosis,as well as accelerating the aging process. Zinc has long been known tohave antioxidant functions. As noted above, various studies foundselenium and zinc at low levels in CLD and HCV-infected patients. Zincand selenium were also found to be decreased in liver cirrhosispatients, and administration of zinc and selenium had positive metaboliceffects on cirrhotic and cancer patients. Vitamin E and selenium werefound to promote hepatic stellate cell apoptosis in rats. Selenium haslong been known for its antioxidant effects, and studies show thatselenium appears to cause up-regulation of manganese superoxidedismutase (MnSOD). Selenium is also involved in the thioredoxin andglutaredoxin thiol-based enzymatic antioxidant systems. Vitamin E andselenium supplied together have been found to decrease hepatic stellatecell activation and hepatic fibrosis.

Vitamin D is a metabolic antioxidant, and it is known to be deficient inall CLD, in particular NAFLD and HCV. The medical food nutrients fulfilla distinctive nutritional requirement for Vitamin D in CLD patients.

The medical food nutrients fulfill a distinctive nutritional requirementfor alpha lipoic acid (ALA), which is a cellular thiol-containingantioxidant ALA can lower oxidative stress and it plays an essentialrole in mitochondrial antioxidant reactions, quenching ROS such assuperoxide radicals, hydroxyl radicals, hypochlorous acid, peroxylradicals, and singlet oxygen. ALA also reduces vitamin C and GSH, whichin turn recycles vitamin E. ALA fulfills a requisite distinctivenutritional need because of the role that ALA plays in CMP-alteredmetabolic systems, especially in CMP-altered cellular redox homeostasis.ALA directly scavenges ROS, but it also recycles other antioxidants likeGSH and vitamin C and prevents toxicities associated with theirdepletion. ALA promotes GSH synthesis and vitamin C levels, andmodulates transcription factors like NFkappaB. Studies show ALA hasdramatic effects in oxidative stress conditions. L-carnitine and ALAreversed mitochondrial oxidative damage and serum liver enzymes in NASHmodel mice. Further, ALA chelates metal ions like iron and copper.

ALA is an important constituent of the linked-chain intracellularantioxidant system and it is known to have potent redox properties, butevidence shows that besides being a direct scavenger of oxidants, ALAhas been shown to stimulate GSH synthesis through an up-regulation of atranscription factor, Nrf2. Nrf2 determines the expression ofantioxidant and detoxification genes regulated by the antioxidantresponse element (ARE). Significantly, ALA has also been shown tomodulate NF-kappaB transcription factor activity, which is involved inhepatic stellate cell activation.

CLD-induced oxidative stress establishes the distinctive nutritionalrequirement for thiol-containing antioxidants as well as non-enzymaticsmall molecule cellular antioxidants, of which ALA is both. The medicalfood medical food protocol provides moderate amounts of ALA to fulfillthe distinctive nutritional requirements of CLD patients.

The Medical Food of the Present Invention Supplies BotanicalPolyphenols—Integral Components of the Body's Physiological AntioxidantResponse

The medical food provides polyphenols from GRAS botanicals due to thenew distinctive nutritional requirements of CLD patients and theirCMP-associated disruption of redox system homeostasis and inducement ofextreme oxidative stress. Polyphenols from GRAS botanicals are importantintegral components of the body's normal metabolic response to oxidativestress. Studies have shown that botanical polyphenols prevent Nrf2translocation and modulate NF-kappaB pathways, thereby protect DNA fromoxidative stress-mediated damage. Dietary botanical polyphenols areintegral components of our bodies' physiological antioxidant response.Administration of botanical polyphenols and other antioxidantmetabolites have been shown to be helpful in liver disease due to theirmetabolic antioxidant effects.

The medical food provides the following GRAS botanicals whoseadministration in animal and human studies on CMP-associated oxidativestress have been demonstrated:

Piper cubeba (cubeb berries) has been used in folk medicine forcenturies. It contains monoterpenes and sesquiterpenes among itsphenolic components while also providing micronutrients. Piper alsocontains piperine, which has been associated with antioxidant efficacy,inhibition of liver fibrosis and hypolipidic effects in high-fat dietrats. Piperine has also been shown to inhibit the macrophageinflammatory response.

Cynara scolymus (artichoke) has been shown to reduce hepatic oxidativestress and restore lipoprotein homeostasis in rats fed a highcholesterol diet. Polyphenols from artichoke have been shown to markedlyreduce hepatic oxidative stress in rats and to prevent the loss of GSHin rat hepatocytes. Further, artichoke was found to inhibit cholesterolbiosynthesis in rat hepatocytes. As seen in Part A, cholesterol levelsincrease in patients with CLD due to CMP-associated metabolic changes.

Nigella sativa (black cumin) administration to HCV patients in Egypt wasfound to be “. . . tolerable, safe, decreased viral load, and improvedoxidative stress, clinical condition and glycemic control in diabeticpatients”. Black cumin seeds and oils have been found to behepatoprotective against hepatotoxicity induced by either disease orchemicals. The beneficial effects are likely related to theircytoprotective and metabolic antioxidant actions. Inlipopolysaccharide-induced inflammation, black cumin has an antioxidanteffect. Black cumin has also been shown to have beneficialimmunomodulatory properties related to its metabolic antioxidantproperties.

Curcuma longa (Turmeric) has been found to have significant hepaticantioxidant properties in ethanol-induced oxidative stress, in rabbitsfed an atherogenic diet, and in liver oxidative damage induced by leadacetate in mice. Turmeric extract (curcumin) has been found to inhibitthe progression of liver cirrhosis in thiocetamide induced livercirrhosis in rats. Turmeric extract was also found to regulate plasmacholesterol and fatty liver in rats fed a high-cholesterol diet Turmericexerts an antioxidative effect on phospholipid peroxidation and hepaticlipid metabolism in mice fed a high cholesterol or atherogenic dietCurcumin has long been shown to be an effective antioxidant nutrient inliver diseases. Further, Curcumin inhibits several factors like nuclearfactor NF-kappaB, a prototypical proinflammatory signaling pathway.Curcumin attenuates liver injury induced by ethanol, thioacetamide, ironoverdose, cholestasis and acute, subchronic and chronic carbontetrachloride (CCl(4)) intoxication. Moreover, curcumin reverses CCl (4)induced cirrhosis. Curcumin has been shown to be hepatoprotectiveagainst ethanol-induced hepatic fibrosis by inhibiting hepatic stellatecell proliferation and by suppressing TGF-Beta signaling.

Grape Seed extract has been shown to reduce oxidative stress inexperimental animal biliary obstruction studies, in methotrexate inducedoxidative stress in rat liver, in radiation induced oxidative stress inrat liver, and in a rat model of diabetes mellitus, which is a conditionthat aggravates CLD. Grape Seed extract has also been shown to improveliver function in patients with NAFLD.

The Medical Food of the Present Invention Supplies Phosphatidylcholine(PC), which fulfills a Distinctive Nutritional Requirement for IncreasedPC in Chronic Liver Disease Patients

A recent review summarized current and historical in-vitro and clinicalstudies on PC. The animal studies and in-vitro studies reviewed in theanalysis found that, “. . . EPL influenced membrane-dependent cellularfunctions and showed anti-oxidant, anti-inflammatory, anti-fibrotic,apoptosis-modulating, regenerative, membrane-repairing and -protective,cell-signaling and receptor influencing, as well as lipid-regulatingeffects in intoxication models with chemicals or drugs.” The review alsoanalyzed clinical studies, where patients with CLD of all etiologies, “.. . have shown improvement in subjective symptoms; clinical, biochemicaland imaging findings; and histology in liver indications such as fattyliver of different origin, drug hepatotoxicity, and adjuvant in chronicviral hepatitis and hepatic coma.

PC is a main cellular membrane phosphoplipid, and it is associated withproper cell membrane and mitochondrial membrane form and function. PC issynthesized in two different biosynthetic pathways in the body. Theseinclude the SAMe dependent PEMT pathway and CDP-choline pathway. In thePEMT pathway, SAMe is involved in three successive methylations ofphosphatidylethanolamine (PE) to form PC. The PEMT pathway occurs in theliver only. The other main PC biosynthetic pathway is called theCDP-choline pathway (or the Kennedy pathway) and it occurs in the restof the cells of the body. In this pathway, PC is created throughconversion of dietary choline into CDP-choline and then to PC.

CLD causes decreased availability of PC, and oxidation of PC inmembranes can occur in CLD due to extreme oxidative stress. It has beenshown that CLD creates increased demand for PC and choline. The medicalfood supplies PC, the biosynthetic end-product of both PC pathways. PCadministration spares SAMe from increased flux into the PEMT PC pathway.PC administration also produces choline, which is produced from thecatabolism of PC. Both dietary choline and PC-derived choline are eithercycled back to the CDP-choline pathway for PC production or cycled tothe production of betaine, a necessary one-carbon donor involved in theSAM/SAH/methionine metabolism, or also to acetylcholine.

Choline regulates cholesterol metabolism, which is another up-regulatedCMP-associated pathway. Choline supplementation improved liver functionand prevented NASH in a study on PEMT knockout mice. As reported by AlRajabi et al, hepatic cholesterol but not triglyceride was normalizedwith a significant improvement in liver function when supplemented withcholine. They concluded that their findings suggested choline canmaintain cholesterol homeostasis and thereby promote liver health.

Dr. Lieber found that oral supplementation of PC caused increased GSHsynthesis due to increased SAMe availability. This increase in SAMestores was due to decreased metabolic demand for SAMe into the PEMTpathway due to PC end-product supplementation. Dr. Leiber surmised, “. .. it is likely that . . . providing PCs, decreases the utilization ofSAMe and thereby contributes to its restoration, with replenishment ofGSH and correction of the alcohol-induced oxidative stress”.

The Lieber study above showed that PC supplementation decreases flux ofSAMe from the PC biosynthetic pathway to the GSH biosynthetic pathway.It is also true that the flux of SAMe may be decreased into the CMPup-regulated GSH biosynthetic pathway if that pathway is supplied withadditional amounts of NAC. NAC and SAMe are the two rate-limitingmetabolites in GSH biosynthesis, and either will promote the productionof GSH. NAC has been shown to decrease oxidative stress in alcoholichepatitis and in cirrhosis animal studies .

Interestingly, PC administration by itself has shown an anti-fibrogeniceffect in studies performed on patients with CLD. A European PC clinicalstudy found reduced levels of procollagen-III-peptide in HBV patients,while a study on HCV patients found decreased levels of albumin-boundhydroxyproline. Remarkably, anti-fibrotic improvement in histology wasshown in pharmacological and clinical studies performed on patients andanimals administered PC.

The Medical Food of the Present Invention Supplies Metabolites of theSAM/SAH/Methionine Cycles, which Experience Greater Utilization byChronic Liver Disease Patients

Studies showed SAMe has positive effects on oxidative stress in CLD,increasing GSH levels, which are depleted in CLD. It is known that SAMemay play an important role in reversing hepatic glutathione depletion inpatients with CLD. While the medical food does not supply SAMe, itsupplies the other rate limiting metabolite of GSH synthesis, NAC, forGSH synthesis instead. Supplying NAC decreases demand for SAMe in theGSH biosynthetic loop because SAMe is converted to NAC for GSHsynthesis. The medical food also supplies PC to the CMP up-regulated PCpathway; thus, decreasing flux of SAMe into the PC pathway. Both ofthese actions free up potential SAMe stores for DNA methylation or themany other biosynthetic functions of SAMe.

A 1989 study showed that SAMe increases hepatic GSH in patients withliver disease. In an ethanol-induced fibrotic mouse model, SAMeadministration was shown to attenuate oxidative stress and hepaticstellate cell activation. SAM/SAH/methionine cycling has been shown tobe impaired in CLD and administration of one-carbon metabolites havebeen shown to restore proper cycling, see Section 5 below. Thus, themedical food contemplated herein fulfills the distinctive nutritionalrequirements for increased SAMe and one-carbon metabolites for properSam/SAH/methionine cycling.

Methionine/homocysteine metabolism and choline metabolism areinterdependent Choline is recycled from catabolized PC and is convertedmainly back to PC, but also to acetylcholine and betaine. Feeding a dietdeficient in choline and methionine has been used as a mechanism tocreate steatosis in the lab for studies. As noted previously, all ofthese CLD-affected pathways are linked to SAM/SAH/methionine and GSHmetabolism, and therefore nutritional deficiencies in any of thesepathways may contribute to oxidative stress.

The Medical Food of the Present Invention Supplies Metabolites ofOne-Carbon Methyl Metabolism

The medical food formula also includes one-carbon methyl donors such asfolate, vitamin B12, vitamin B6 and betaine. IncreasedSAM/SAH/methionine cycling creates greater demand for one-carbonmethylation factors. These one-carbon metabolites are distinctiverequired nutrients provided in the medical food medical food protocolfor their one-carbon methyl donating properties and their interlinkedduties with the SAM/SAH/methionine cycle and the homocysteine/NAC/GSHcycle, both of which experience greater utilization in patients withCLD.

One-carbon methyl donors like folate, vitamin B12 and betaine converthomocysteine to methionine, and vitamin B6 directs homocysteine to NAC,which is then ultimately converted to GSH. Proper methylation by theseone-carbon methyl donors is important to avoid accumulation ofhomocysteine in the body. Therefore, any up-regulation of theSAM/SAH/methionine cycle must necessarily be accompanied by increasedutilization of these critical one-carbon methyl donors.

A study showed that ALD patients experience decreased folate, vitamin B6and thiamine levels, suggesting that these deficiencies were due to thepatients' inability to absorb those vitamins from food. Interestingly,these patients were able to absorb synthesized supplemental forms ofthese vitamins, in spite of their inability to absorb the nutrients fromfood. Another study showed Thiamine supplementation reversedethanol-induced hepatotoxicity in rats.

Betaine, one of a necessary medical food metabolites, aids in theconversion of homocysteine to methionine. Betaine is also athiol-enhancing cofactor.

One-carbon methyl donors have positive nutritional effects on redoxhomeostasis and oxidative stress due to their intrinsic role inSAM/SAH/methionine metabolism. As noted previously, the medical fooddoes not provide SAMe or methionine to satisfy the distinctivenutritional requirement of CLD patients for increased SAMe. Rather, themedical food concentrates on saving SAMe by decreasing the draw of SAMeinto the other highly up-regulated CMP pathways, the PC and GSHpathways. Further, SAMe-homocysteine-methionine cycling is impaired dueto decreased enzymatic activity, which the medical food restores byadministering one-carbon metabolic methyl donors like betaine, whichprotects against ethanol-induce fatty liver infiltration in ALD.Betaine's restorative powers to disrupted ALD metabolic pathways isgenerally attributed to its role in restoring SAMe supplies and therebydecreasing oxidative stress. A study conducted by Yung et al documented,that betaine's hepatoprotective activity is associated with its effectson sulfur amino acid metabolism.

The Medical Food of the Present Invention Supplies Metabolites ofCLD-Depleted Amino Acid Metabolism

CLD increases utilization and decreases availability of L-Carnitine.L-Carnitine levels are diminished in all CLD, in particular HCV, andthis makes it a required metabolite in the medical food medical foodformula. Depletion of L-Carnitine may have negative effects onCLD-associated steatosis because L-Carnitine transports cytosolic fattyacids to the mitochondria for β-oxidation. β-oxidation of fatty acids isanother CMP up-regulated activity. The medical food supplies L-lysine,which combines with methionine to form the CMP-depleted amino acid,L-Carnitine. The medical food also supplies L-Carnitine to meetincreased demands for increased β-oxidation of fatty acids in patientswith CLD. In a NASH mouse model, “L-Carnitine prevents progression ofNASH in a mouse model by up-regulating the mitochondrial β-oxidation andredox system”. L-Carnitine and ALA reversed mitochondrial oxidativedamage and serum liver enzymes in NASH mouse model.

The medical food also supplies arginine as a required amino acidmetabolite in CLD patients. Arginine is oxidized to form nitric oxide(NO). NO is involved in the modulation of hepatic microcirculatoryperfusion and oxygenation in cholesterol-induced hepatic steatosis.Arginine administration selectively increases NO levels, which improveshepatic microcirculation and tissue oxygenation in patients withcirrhosis. A study conducted by Nanji et al reported that “. . . ourresults show that arginine administration, probably through thegeneration of nitric oxide, leads to improvement in pathological changessuch as fatty liver, necrosis, inflammation, and fibrosis. Theseimprovements were accompanied by down-regulation of nuclear factorNF-κB, pro-inflammatory cytokines cyclooxygenase-2, and inducible nitricoxide synthase”. GSH depletion leads to NO toxicity, so the medical foodcontemplated herein supplies the GSH metabolites NAC, L-glutamate andL-lysine for GSH production.

The medical food supplies arginine as a necessary amino acid metabolitefor patients with CLD. Decreased arginine levels may be associated withhepatic encephalopathy and hyperammonemia due to urea cycle disruption.Studies have found that the CMP-associated metabolic disruption of theof ammonia detoxification pathway, “. . . preceed the histologicalmanifestation of irreversible liver damage”. Arginine administrationmust be balanced with lysine administration, which is also present inthe medical food formula. Lysine is also a metabolic precursor ofL-carnitine, as mentioned above.

Exemplary Composition Embodiments

In a particular embodiment, a daily dose of the medical foodcontemplated herein may comprise the following ingredients in thefollowing mass ranges:

L-Lysine in a range of 400-5,000 mg

Cysteine-providing ingredient (such as NAC or L-cysteine) in a range of500-5,000 mg

L-Arginine in a range of 1,000-9,000 mg

Polyenylphosphatidylcholine in a range of 500-10,000 mg

Alpha Lipoic Acid in a range of 200-2,500 mg

Vitamin C (as ascorbic acid & calcium ascorbate) in a range of500-10,000 mg

N-Acetyl L-Carnitine in a range of 250-3,000 mg

Betaine HCl in a range of 300-20,000 mg

L-Glutamate in a range of 200-2,000 mg

Turmeric (Curcuma longa) in a range of 200-1,500 mg

Grape seed extract (95% Proanthocyanidins) in a range of 100-1,000 mg

Black cumin seed (Nigella sativa) in a range of 50-400 mg

Pantothenic acid (as calcium pantothenate) in a range of 20-10,000 mg

Magnesium (as magnesium citrate) in a range of 50-800 mg

Vitamin E (as mixed tocopherols) in a range of 50-1,000 IU

Artichoke (leaf) extract (Cynara scolymus) in a range of 25-300 mg

L-Glycine in a range of 50-3000 mg

Vitamin B1 in a range of 10-200 mg

Vitamin B2 in a range of 10-200 mg

CoQ10 (as ubiquinol) in a range of 30-1,000 mg

Cubeb berries (Piper cubeba) in a range of 10-100 mg

Vitamin B3 in a range of 45-3,000 mg

Vitamin B6 (as pyridoxine HCl) in a range of 10-200 mg

Zinc (as zinc citrate) in a range of 5-50 mg

Vitamin D3 in a range of 400-10,000 IU

Folate (as folic acid) in a range of 200-3,000 mcg

Vitamin B12 (as methylcobalamin) in a range of 200-3,000 mcg

Selenium (as selenate aspartate) in a range of 100-600 mcg

Vitamin A in a range of 200-3,000 μg retinol activity equivalents

Vitamin K in a range of 30-5,000 mcg

Biotin in a range of 50-2,000 mcg

Optionally, cannabidiol in a range of 2.5-1,500 mg.

It should be understood that in varying embodiments, the present medicalfood may not have all of the above listed components. Indeed many may beomitted. In a particular embodiment, any combination of aCysteine-providing ingredient, Alpha Lipoic Acid,Polyenylphosphatidylcholine, and one or more of the above listedcomponents may constitute the medical food of the present invention. Inanother embodiment, the medical food the composition may compriseCysteine-providing ingredient, Alpha Lipoic Acid,Polyenylphosphatidylcholine.

In still another embodiment a daily dose of the medical foodcontemplated herein may comprise the following ingredients in thefollowing mass ranges:

Cysteine providing ingredient such as N-Acetyl Cysteine or L-Cysteine ina range of 500-5,000 mg

L-Arginine in a range of 1,000-9,000 mg

Polyenylphosphatidylcholine in a range of 500-10,000 mg

Alpha Lipoic Acid in a range of 200-2,500 mg

Turning now to FIG. 1, a flow chart of an embodiment of use of themedical food of the present invention is provided. In this view,initially a patient having chronic liver disease is identified. Thispatient is then provided with a three times daily regimen of anembodiment of the medical food. The medical food may be taken in anynumber of manners, as noted above. After a period of time with takingthe three times daily regimen of medical food, the patient may be testedto track progress and efficacy. This testing may involve any sort ofsampling, including urine, blood, liver, fat, and/or tissue sampling,and the like. Based on the changes measured and caused by the medicalfood regimen, it may be adjusted, continued, or the like.

While several variations of the present invention have been illustratedby way of example in preferred or particular embodiments, it is apparentthat further embodiments could be developed within the spirit and scopeof the present invention, or the inventive concept thereof. However, itis to be expressly understood that such modifications and adaptationsare within the spirit and scope of the present invention, and areinclusive, but not limited to the following appended claims as setforth.

What is claimed is: 1) A method of reducing liver fibrosis comprisingthe steps of administering a composition to a patient, the compositioncomprising: a cysteine-providing ingredient in a range of 500-5,000 mg;polyenylphosphatidylcholine in a range of 500-10,000 mg; alpha lipoicacid in a range of 200-2,500 mg; and at least one of: L-lysine in arange of 400-5,000 mg; L-arginine in a range of 1,000-9,000 mg; vitaminC in a range of 500-10,000 mg; N-acetyl L-carnitine in a range of250-3,000 mg; betaine HCl in a range of 300-20,000 mg; L-glutamate in arange of 200-2,000 mg; turmeric in a range of 200-1,500 mg;proanthocyanidins in a range of 100-1,000 mg; nigella sativa in a rangeof 50-400 mg; pantothenic acid in a range of 20-10,000 mg; magnesium ina range of 50-800 mg; vitamin E in a range of 50-1,000 IU; cynarascolymus in a range of 25-300 mg; L-glycine in a range of 50-3000 mg;vitamin B1 in a range of 10-200 mg; vitamin B2 in a range of 10-200 mg;ubiquinol in a range of 30-1,000 mg; piper cubeba in a range of 10-100mg; vitamin B3 in a range of 45-3,000 mg; vitamin B6 in a range of10-200 mg; zinc in a range of 5-50 mg; vitamin D3 in a range of400-10,000 IU; folate in a range of 200-3,000 mcg; vitamin B12 in arange of 200-3,000 mcg; selenium in a range of 100-600 mcg; vitamin A ina range of 200-3,000m retinol activity equivalents; Vitamin K in a rangeof 30-5,000 mcg; and biotin in a range of 50-2,000 mcg; wherein theadministering is repeated every 24 hours; and testing the patient, thestep of testing comprising taking a sample from the patient, analyzingthe sample for an indicator of liver fibrosis, and comparing theanalyzed sample to a previously analyzed sample. 2) The method of claim1 wherein the composition comprises each of: L-lysine in a range of400-5,000 mg; L-arginine in a range of 1,000-9,000 mg; vitamin C in arange of 500-10,000 mg; N-acetyl L-carnitine in a range of 250-3,000 mg;betaine HCl in a range of 300-20,000 mg; L-glutamate in a range of200-2,000 mg; turmeric in a range of 200-1,500 mg; proanthocyanidins ina range of 100-1,000 mg; nigella sativa in a range of 50-400 mg;pantothenic acid in a range of 20-10,000 mg; magnesium in a range of50-800 mg; vitamin E in a range of 50-1,000 IU; cynara scolymus in arange of 25-300 mg; L-glycine in a range of 50-3000 mg; vitamin B1 in arange of 10-200 mg; vitamin B2 in a range of 10-200 mg; ubiquinol in arange of 30-1,000 mg; piper cubeba in a range of 10-100 mg; vitamin B3in a range of 45-3,000 mg; vitamin B6 in a range of 10-200 mg; zinc in arange of 5-50 mg; vitamin D3 in a range of 400-10,000 IU; folate in arange of 200-3,000 mcg; vitamin B12 in a range 200-3,000 mcg; seleniumin a range of 100-600 mcg; vitamin A in a range of 200-3,000m retinolactivity equivalents; Vitamin K in a range of 30-5,000 mcg; and biotinin a range of 50-2,000 mcg. 3) The method of claim 1 further comprisingthe step of dividing the composition into a plurality of capsules, eachof the plurality of capsules comprising a fraction of the composition.4) The method of claim 1 wherein the testing is selected to analyze arate of one carbon methylation metabolism which is supported bynutrients provided in the administration step. 5) The method of claim 1wherein the testing is selected to analyze a rate of S-adenosylmethionine metabolism which is supported by nutrients provided in theadministration step. 6) The method of claim 1 wherein the step ofadministration comprises administering a powdered composition. 7) Themethod of claim 1 wherein the cysteine-providing ingredient isL-cysteine. 8) The method of claim 1 wherein the testing furthercomprises analyzing a metabolic marker. 9) The method of claim 8 whereinthe metabolic marker is phosphatidylcholine homeostasis. 10) The methodof claim 8 wherein the metabolic marker is a lysophosphatidylcholinespecies level. 11) The method of claim 1 wherein the composition furthercomprises cannabidiol in a range of 2.5-1,500 mg. 12) The method ofclaim 11 wherein the composition is selected to supply metabolitesinvolved in glutathione biosynthesis. 13) The method of claim 1 whereinthe composition is selected to provide a full complement of interlinkedsmall-molecule non-enzymatic antioxidants, including the antioxidantminerals zinc, selenium, magnesium, CoQ10, vitamins A, B, C, D, E, andthe thiol-based cellular antioxidant ALA. 14) The method of claim 1further comprising the step of orally administering the composition. 15)A method of treatment for a patient having chronic liver diseasecomprising the steps of: identifying that the patient suffers fromchronic liver disease based on a testing of a metabolic marker;administering a composition selected to reduce oxidative stress causedby an adjusted nutritional requirement caused by the chronic liverdisease, the composition comprising: polyenylphosphatidylcholine in arange of 500-10,000 mg; alpha lipoic acid in a range of 200-2,500 mg;and at least one of: L-lysine in a range of 400-5,000 mg; L-arginine ina range of 1,000-9,000 mg; vitamin C in a range of 500-10,000 mg;N-acetyl L-carnitine in a range of 250-3,000 mg; betaine HCl in a rangeof 300-20,000 mg; L-glutamate in a range of 200-2,000 mg; turmeric in arange of 200-1,500 mg; proanthocyanidins in a range of 100-1,000 mg;nigella sativa in a range of 50-400 mg; pantothenic acid in a range of20-10,000 mg; magnesium in a range of 50-800 mg; vitamin E in a range of50-1,000 IU; cynara scolymus in a range of 25-300 mg; L-glycine in arange of 50-3000 mg; vitamin B1 in a range of 10-200 mg; vitamin B2 in arange of 10-200 mg; ubiquinol in a range of 30-1,000 mg; piper cubeba ina range of 10-100 mg; vitamin B3 in a range of 45-3,000 mg; vitamin B6in a range of 10-200 mg; zinc in a range of 5-50 mg; vitamin D3 in arange of 400-10,000 IU; folate in a range of 200-3,000 mcg; vitamin B12in a range of 200-3,000 mcg; selenium in a range of 100-600 mcg; vitaminA in a range of 200-3,000 μg retinol activity equivalents; Vitamin K ina range of 30-5,000 mcg; and biotin in a range of 50-2,000 mcg; whereinthe administering step is repeated every 24 hours; and testing thepatient, the step of testing comprising taking a sample from thepatient, analyzing the sample for the metabolic marker, and comparingthe analyzed sample to a previously analyzed sample of the metabolicmarker. 16) The method of claim 15 wherein the composition compriseseach of: L-lysine in a range of 400-5,000 mg; L-arginine in a range of1,000-9,000 mg; vitamin C in a range of 500-10,000 mg; N-acetylL-carnitine in a range of 250-3,000 mg; betaine HCl in a range of300-20,000 mg; L-glutamate in a range of 200-2,000 mg; turmeric in arange of 200-1,500 mg; proanthocyanidins in a range of 100-1,000 mg;nigella sativa in a range of 50-400 mg; pantothenic acid in a range of20-10,000 mg; Magnesium in a range of 50-800 mg; Vitamin E in a range of50-1,000 IU; cynara scolymus in a range of 25-300 mg; L-glycine in arange of 50-3000 mg; vitamin B1 in a range of 10-200 mg; vitamin B2 in arange of 10-200 mg; ubiquinol in a range of 30-1,000 mg; piper cubeba ina range of 10-100 mg; vitamin B3 in a range of 45-3,000 mg; vitamin B6in a range of 10-200 mg; Zinc in a range of 5-50 mg; vitamin D3 in arange of 400-10,000 IU; folate in a range of 200-3,000 mcg; vitamin B12in a range of 200-3,000 mcg; selenium in a range of 100-600 mcg; vitaminA in a range of 200-3,000 μg retinol activity equivalents; Vitamin K ina range of 30-5,000 mcg; and biotin in a range of 50-2,000 mcg. 17) Themethod of claim 15 wherein the composition further comprises L-cysteine.18) The method of claim 15 wherein the metabolic marker is at least oneof phosphatidylcholine homeostasis and a lysophosphatidylcholine specieslevel. 19) The method of claim 15 wherein the composition furthercomprises cannabidiol in a range of 2.5-1,500 mg. 20) The method ofclaim 15 wherein the step of testing comprising taking a blood samplefrom the patient, analyzing the sample for appropriate liver functionusing blood tests, and comparing results of the analysis to a blood testfrom the patent analyzed before a first administration step.